Fig. 2: USP4 interacts with both BRCA1 and BARD1.
From: The deubiquitinating enzyme USP4 regulates BRCA1 stability and function
a USP4 interacts with both BRCA1 and BARD1. 293 T cells were transfected with the indicated plasmids, and Flag-USP4 IPs were performed. BRCA1 and BARD1 were examined by WB using antibodies against HA or myc. b Endogenous USP4 interacts with BRCA1 in MCF-10A cells. Anti-USP4 antibody was used for IP and the blot was probed with anti-USP4 or BRCA1 antibody, respectively. c Endogenous BRCA1 interacts with USP4 in MCF-10A cells. Endogenous BRCA1 was pulled down by anti-BRCA1 antibody and the IPs was blotted with anti-BRCA1 or USP4 antibody, respectively. d Interaction of USP4 with BRCA1 upon treatment of DNA damage agent. HEK293T cells were treated with the indicated DNA damage agents and the IPs of USP4 were analyzed by WB with the indicated antibodies. BRCA1 pulled down by USP4 was quantified and normalized with that of control (No treatment). e Interaction between USP4 and BRCA1 during cell cycle. HeLa cells were synchronized to G1/S boundary by double-thymidine block, and then released for the indicated time. The cell lysates of G1, S and G2/M phase were subjected to immunoprecipitation using anti-USP4 antibody. The IPs were analyzed by WB with the indicated antibodies. BRCA1 pulled down by USP4 was quantified and normalized with that of G1/S. Cell cycle was analyzed by FACS, and the distribution of cell cycle was indicated. Asyn, asynchronous cells. f USP4 interacts with BRCA1. HEK293T cells transfected with BRCA1 were lysed and the lysates were incubated with either GST or GST-USP4. GST-pulldowns were blotted with the indicated antibodies. g HA-tagged full-length or truncated BRCA1 were co-expressed with Flag-tagged USP4 in HEK293T cells. Extracts were subjected to IP with anti-Flag-M2 antibody, and analyzed by WB. h Schematic diagram showing the constructs expressing full-length BRCA1 and its truncated fragments (Top panel), or the structure of USP4 and deletion constructs used (bottom panel). i HA-tagged USP4 constructs were co-expressed with Flag-tagged BRCA1 in HEK293T cells. Cellular extracts were subjected to IP with anti-Flag-M2 antibody, and analyzed by WB.
